Targeting Hedgehog Signaling with Glasdegib in Patients with Refractory Sclerotic Chronic GVHD: A Report of Two Phase I/II Trials.

PURPOSE: Sclerotic chronic GVHD (scGVHD) is characterized by progressive skin fibrosis and frequent refractoriness to available therapies. Aberrant activation of Hedgehog signaling in dermal fibroblasts has been implicated in scGVHD. Here, we report the results of two phase I/II studies (NCT03415867, GETH-TC; NCT04111497, FHD) that evaluated glasdegib, a smoothened antagonist, as a novel therapeutic agent in refractory scGVHD. PATIENTS AND METHODS: Adult patients with active scGVHD after ≥1 (FHD) or ≥2 (GETH-TC) lines of therapy were enrolled. Primary endpoints were dose-limiting toxicity (DLT) and MTD in the GETH-TC trial, and safety and tolerability measures in the FHD trial. Glasdegib was administered once daily in 28-day cycles. Responses were scored per 2014 NIH cGVHD criteria. Correlative studies were performed to evaluate the role of fibroblast-independent immune mechanisms on clinical activity. RESULTS: Twenty (GETH-TC) and 15 (FHD) patients were recruited. Treatment-emergent grade (G) ≥2 adverse events (AE) in the GETH-TC trial included muscle cramps (85%), alopecia (50%), and dysgeusia (35%). Two patients experienced a DLT (G3 muscle cramps), and the MTD was established at 50 mg. G3 muscle cramps were the most frequently reported AE (33%) in the FHD trial. At 12-months, the skin/joint scGVHD overall response rate was 65% (all partial responses) in the GETH-TC trial and 47% (6 partial responses, 1 complete response) in the FHD cohort. No immune correlates of response were identified. CONCLUSIONS: Glasdegib demonstrated promising responses in patients with refractory scGVHD, but tolerability was limited by muscle cramping.

Monitoring of kinetics and exhaustion markers of circulating CAR-T cells as early predictive factors in patients with B-cell malignancies.

Purpose: CAR-T cell therapy has proven to be a disruptive treatment in the hematology field, however, less than 50% of patients maintain long-term response and early predictors of outcome are still inconsistently defined. Here, we aimed to optimize the detection of CD19 CAR-T cells in blood and to identify phenotypic features as early biomarkers associated with toxicity and outcomes. Experimental design: In this study, monitoring by flow cytometry and digital PCR (dPCR), and immunophenotypic characterization of circulating CAR-T cells from 48 patients treated with Tisa-cel or Axi-cel was performed. Results: Validation of the flow cytometry reagent for the detection of CAR-T cells in blood revealed CD19 protein conjugated with streptavidin as the optimal detection method. Kinetics of CAR-T cell expansion in blood confirmed median day of peak expansion at seven days post-infusion by both flow cytometry and digital PCR. Circulating CAR-T cells showed an activated, proliferative, and exhausted phenotype at the time of peak expansion. Patients with increased expansion showed more severe CRS and ICANs. Immunophenotypic characterization of CAR-T cells at the peak expansion identified the increased expression of co-inhibitory molecules PD1 and LAG3 and reduced levels of the cytotoxicity marker CD107a as predictors of a better long-term disease control. Conclusions: These data show the importance of CAR-T cells in vivo monitoring and identify the expression of PD1LAG3 and CD107a as early biomarkers of long-term disease control after CAR-T cell therapy.

Combined treatment of graft versus host disease using donor regulatory T cells and ruxolitinib.

Donor derived regulatory T lymphocytes and the JAK1/2 kinase inhibitor ruxolitinib are currently being evaluated as therapeutic options in the treatment of chronic graft versus host disease (cGvHD). In this work, we aimed to determine if the combined use of both agents can exert a synergistic effect in the treatment of GvHD. For this purpose, we studied the effect of this combination both in vitro and in a GvHD mouse model. Our results show that ruxolitinib favors the ratio of thymic regulatory T cells to conventional T cells in culture, without affecting the suppressive capacity of these Treg. The combination of ruxolitinib with Treg showed a higher efficacy as compared to each single treatment alone in our GvHD mouse model in terms of GvHD incidence, severity and survival without hampering graft versus leukemia effect. This beneficial effect correlated with the detection in the bone marrow of recipient mice of the infused donor allogeneic Treg after the adoptive transfer.

FANCD2 maintains replication fork stability during misincorporation of the DNA demethylation products 5-hydroxymethyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxyuridine.

Fanconi anemia (FA) is a rare hereditary disorder caused by mutations in any one of the FANC genes. FA cells are mainly characterized by extreme hypersensitivity to interstrand crosslink (ICL) agents. Additionally, the FA proteins play a crucial role in concert with homologous recombination (HR) factors to protect stalled replication forks. Here, we report that the 5-methyl-2'-deoxycytidine (5mdC) demethylation (pathway) intermediate 5-hydroxymethyl-2'-deoxycytidine (5hmdC) and its deamination product 5-hydroxymethyl-2'-deoxyuridine (5hmdU) elicit a DNA damage response, chromosome aberrations, replication fork impairment and cell viability loss in the absence of FANCD2. Interestingly, replication fork instability by 5hmdC or 5hmdU was associated to the presence of Poly(ADP-ribose) polymerase 1 (PARP1) on chromatin, being both phenotypes exacerbated by olaparib treatment. Remarkably, Parp1-/- cells did not show any replication fork defects or sensitivity to 5hmdC or 5hmdU, suggesting that retained PARP1 at base excision repair (BER) intermediates accounts for the observed replication fork defects upon 5hmdC or 5hmdU incorporation in the absence of FANCD2. We therefore conclude that 5hmdC is deaminated in vivo to 5hmdU, whose fixation by PARP1 during BER, hinders replication fork progression and contributes to genomic instability in FA cells.

Lesión pulmonar asociada al uso de cigarrillos electrónicos o productos de vapeo (EVALI): a propósito de un caso relacionado a neumonía eosinofílica aguda / Lung injury associated with the use of electronic cigarettes or vaping products (EVALI): Apropos of a case related to acute eosinophilic pneumonia

La lesión pulmonar asociada al uso de cigarrillos electrónicos o vapeo (EVALI) es una enfermedad respiratoria aguda o subaguda que puede ser grave y potencialmente mortal. Vapear es el proceso de inhalar un aerosol creado al calentar una sustancia como la nicotina o el tetrahidrocannabinol (THC) con un dispositivo electrónico que funciona con baterías, como un cigarrillo electrónico. En el mundo se han descrito más de 2.000 casos, 2/3 hombres, entre los 13 y 75 años. A continuación, presentamos un caso de una paciente de 15 años, con antecedente de consumo de cigarrillo y cannabis vapeado, quien es llevada por sus padres a una institución de alta complejidad por un cuadro respiratorio agudo grave, descartando inicialmente infección por SARS-CoV-2, en quien finalmente se confirma una EVALI manifestada histológicamente como neumonía eosinofílica aguda.

Vaping or electronic cigarettes (E-cigarette vaping) can induce acute or subacute lung pulmonary which can be serious and potentially life-threatening. Vaping is the process by which a substance such as nicotine or tetrahydrocannabinol (THC), is heated creating aerosols that are inhaled using different devices such as E-cigarette that work on batteries. In the world, there are more than 2000 cases described of which two-thirds are males between the ages of 13 and 75 years of age. Here we present the case of a 15-year-old female patient, with prior use of cigarettes and vaping of cannabis, that is brought to the emergency room by her parents with acute respiratory complains during de COVID 19 pandemic and was finally diagnosed as acute eosinophilic pneumonia as a histological manifestation of EVALI.

Multivalent Calixarene-Based Liposomes as Platforms for Gene and Drug Delivery.

The formation of calixarene-based liposomes was investigated, and the characterization of these nanostructures was carried out using several techniques. Four amphiphilic calixarenes were used. The length of the hydrophobic chains attached to the lower rim as well as the nature of the polar group present in the upper rim of the calixarenes were varied. The lipid bilayer was formed with one calixarene and with the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE. The cytotoxicity of the liposomes for various cell lines was also studied. From the results obtained, the liposomes formed with the least cytotoxic calixarene, (TEAC12)4, were used as nanocarriers of both nucleic acids and the antineoplastic drug doxorubicin, DOX. Results showed that (TEAC12)4/DOPE/p-EGFP-C1 lipoplexes, of a given composition, can transfect the genetic material, although the transfection efficiency substantially increases in the presence of an additional amount of DOPE as coadjuvant. On the other hand, the (TEAC12)4/DOPE liposomes present a high doxorubicin encapsulation efficiency, and a slow controlled release, which could diminish the side effects of the drug.

Metallo-Liposomes of Ruthenium Used as Promising Vectors of Genetic Material.

Gene therapy is a therapeutic process consisting of the transport of genetic material into cells. The design and preparation of novel carriers to transport DNA is an important research line in the medical field. Hybrid compounds such as metallo-liposomes, containing a mixture of lipids, were prepared and characterized. Cationic metal lipids derived from the [Ru(bpy)3]2+ complex, RuC11C11 or RuC19C19, both with different hydrophobic/lipophilic ratios, were mixed with the phospholipid DOPE. A relation between the size and the molar fraction α was found and a multidisciplinary study about the interaction between the metallo-liposomes and DNA was performed. The metallo-liposomes/DNA association was quantified and a relationship between Kapp and α was obtained. Techniques such as AFM, SEM, zeta potential, dynamic light scattering and agarose gel electrophoresis demonstrated the formation of lipoplexes and showed the structure of the liposomes. L/D values corresponding to the polynucleotide's condensation were estimated. In vitro assays proved the low cell toxicity of the metallo-liposomes, lower for normal cells than for cancer cell lines, and a good internalization into cells. The latter as well as the transfection measurements carried out with plasmid DNA pEGFP-C1 have demonstrated a good availability of the Ru(II)-based liposomes for being used as non-toxic nanovectors in gene therapy.

Measurable Residual Disease Assessed by Flow-Cytometry Is a Stable Prognostic Factor for Pediatric T-Cell Acute Lymphoblastic Leukemia in Consecutive SEHOP Protocols Whereas the Impact of Oncogenetics Depends on Treatment.

Robust and applicable risk-stratifying genetic factors at diagnosis in pediatric T-cell acute lymphoblastic leukemia (T-ALL) are still lacking, and most protocols rely on measurable residual disease (MRD) assessment. In our study, we aimed to analyze the impact of NOTCH1, FBXW7, PTEN, and RAS mutations, the measurable residual disease (MRD) levels assessed by flow cytometry (FCM-MRD) and other reported risk factors in a Spanish cohort of pediatric T-ALL patients. We included 199 patients treated with SEHOP and PETHEMA consecutive protocols from 1998 to 2019. We observed a better outcome of patients included in the newest SEHOP-PETHEMA-2013 protocol compared to the previous SHOP-2005 cohort. FCM-MRD significantly predicted outcome in both protocols, but the impact at early and late time points differed between protocols. The impact of FCM-MRD at late time points was more evident in SEHOP-PETHEMA 2013, whereas in SHOP-2005 FCM-MRD was predictive of outcome at early time points. Genetics impact was different in SHOP-2005 and SEHOP-PETHEMA-2013 cohorts: NOTCH1 mutations impacted on overall survival only in the SEHOP-PETHEMA-2013 cohort, whereas homozygous deletions of CDKN2A/B had a significantly higher CIR in SHOP-2005 patients. We applied the clinical classification combining oncogenetics, WBC count and MRD levels at the end of induction as previously reported by the FRALLE group. Using this score, we identified different subgroups of patients with statistically different outcome in both Spanish cohorts. In SHOP-2005, the FRALLE classifier identified a subgroup of high-risk patients with poorer survival. In the newest protocol SEHOP-PETHEMA-2013, a very low-risk group of patients with excellent outcome and no relapses was detected, with borderline significance. Overall, FCM-MRD, WBC count and oncogenetics may refine the risk-stratification, helping to design tailored approaches for pediatric T-ALL patients.

Genotoxicity of tetrahydrofolic acid to hematopoietic stem and progenitor cells.

Metabolically reactive formaldehyde is a genotoxin and a carcinogen. Mice lacking the main formaldehyde-detoxifying gene Adh5 combined with the loss of the Fanconi anemia (FA) DNA repair pathway rapidly succumbed to bone marrow failure (BMF) primarily due to the extensive ablation of the hematopoietic stem cell (HSC) pool. However, the mechanism by which formaldehyde mediates these toxic effects is still unknown. We uncover a detrimental role of tetrahydrofolic acid (THF) in cells lacking Adh5 or the FA repair pathway. We show that Adh5- or FA-deficient cells are hypersensitive to formaldehyde and to THF, presenting DNA damage and genome instability. THF cytotoxicity involved imbalance of the nucleotide pool by deregulation of the thymidylate synthase (TYMS) enzyme, which stalled replication forks. In mice, THF exposure had widespread effects on hematopoiesis, affecting the frequency and the viability of myeloid- and lymphoid-committed precursor cells. Moreover, the hematopoietic stem and progenitor cells (HSPC) showed genomic instability, reduced colony-forming capacity and increased frequency of cycling and apoptotic HSCs upon THF exposure. Overall, our data reveal that the physiological pool of THF and formaldehyde challenge the stability of the genome of HSPCs that might lead to blood disorders.

Kon-tiki enhances PS2 integrin adhesion and localizes its ligand, Thrombospondin, in the myotendinous junction.

Cell-extracellular-matrix adhesion is mediated by cell receptors, mainly integrins and transmembrane proteoglycans, which can functionally interact. How these receptors are regulated and coordinated is largely unknown. We show that the conserved transmembrane Drosophila proteoglycan Kon-tiki (Kon, also known as Perdido) interacts with the αPS2ßPS integrin (αPS2 is encoded by inflated and ßPS by myospheroid) to mediate muscle-tendon adhesion. kon and inflated double mutant embryos show a synergistic increase in muscle detachment. Furthermore, Kon modulates αPS2ßPS signaling at the muscle attachment, since phosphorylated Fak is reduced in kon mutants. This reduction in integrin signaling can be rescued by the expression of a truncated Kon protein containing its transmembrane and extracellular domains, suggesting that these domains are sufficient to mediate this signaling. We show that these domains are sufficient to properly localize the αPS2ßPS ligand, Thrombospondin, to the muscle attachment, and to partially rescue Kon-dependent muscle-tendon adhesion. We propose that Kon can engage in a protein complex with αPS2ßPS and enhance integrin-mediated signaling and adhesion by recruiting its ligand, which would increase integrin-binding affinity to the extracellular matrix, resulting in the consolidation of the myotendinous junction.

P-Sulfocalix[6]arene as Nanocarrier for Controlled Delivery of Doxorubicin.

Given the high toxicity of the anthracycline antibiotic doxorubicin (DOX), it is relevant to search for nanocarriers that decrease the side effects of the drug and are able to transport it towards a therapeutic target Here, the encapsulation of DOX by p-sulfocalix[6]arene (calix) has been studied. The interaction of DOX with the macrocycle, as well as with DNA, has been investigated and the equilibrium constant for each binding process estimated. The results showed that the binding constant of DOX to DNA, KDNA , is three orders of magnitude higher than that to calix, Kcalix . The ability of calixarenes to encapsulate DOX molecules, as well as the capability of the DOX molecules included into the inner cavity of the macrocycle to bind with DNA have been examined. Cytotoxicity measurements were done in different cancer and normal cell lines to probe the decrease in the toxicity of the encapsulated DOX. The low toxicity of calixarenes has also been demonstrated for different cell lines.

Eosinófilos en sangre y en esputo como predictores de severidad en crisis asmática / Eosinophils in blood and in sputum as predictors of severity in asthmatic crises

Algunos estudios sugieren que los eosinófilos se han correlacionado con mayor severidad, peor control del asma, peores índices de función pulmonar, mayor hiperreactividad bronquial medida con test de metacolina. No obstante, desconocemos el comportamiento de esta correlación en Colombia y en la clinica de asma del Hospital San Ignacio. Actualmente el wright de esputo se interpreta solo a la luz de la presencia de neutrófilos ignorando el valor de los eosinófilos. Métodos: se realizó un estudio observacional descriptivo y de concordancia, durante un año, en personas mayores de 15 años, que ingresaron por urgencias con crisis asmática requirieron hospitalización. Se determinó en ellos la severidad de la crisis, la severidad de los eosinófilos en sangre y en esputo, el pico espiratorio flujo, los días de estancia totales, en piso, o en UCI y la necesidad de ventilación mecánica para definir si había correlación entre las severidades de estas variables como predictores de severidad de la crisis asmática. Resultados: se incluyeron un total de 169 pacientes, con promedio de edad de 43,3 años, predominantemente mujeres (84%) de los cuales el 56,8% tenían crisis asmática severa y 43,2% moderada. Con un promedio de eosinófilos en esputo de 17,2% y en sangre de 375,5 células por metro cúbico. Promedio de estancia hospitalaria total de 4,7 días, 0,5 días en UCI (solamente 20 pacientes requirieron UCI), y 0,2 días en ventilación mecánica. La mayoría de pacientes tenían eosinófilos en sangre dentro de los valores normales o levemente aumentados y la mayoría del grupo tenía eosinofilia leve en esputo. El PEF al ingreso correspondía en promedio a un 40% del mejor. Solamente se observó concordancia leve entre eosinófilos en esputo y eosinófilos en sangre. No se encontró correlación entre severidad según eosinófilos en esputo o en sangre y los días de estancia, el PEF ni con los medicamentos recibidos. Conclusión: no existe correlación entre los niveles de eosinofilia en sangre o en esputo y la severidad de la crisis medida en días de estancia, dias de ventilación mecánica o valor del pico espiratorio flujo.

Uso clínico de las pruebas de función pulmonar y la tomografía de alta resolución en las enfermedades pulmonares intersticiales / Clinical use of pulmonary function tests and high-resolution tomography ininterstitial lung diseases

En las enfermedades pulmonares intersticiales (EPID) la historia clínica, el examen físico y las imágenes, especialmente la tomografía de alta resolución (TACAR), por lo general, establecen un diagnóstico. Es importante mencionar que aunque este grupo de enfermedades tienen diferentes características clínicas e histológicas; comparten una patrón básico de anomalías de la función pulmonar. En el caso de la TACAR, estas enfermedades tienen características similares, pero hay diferencias puntuales que ayudan en el abordaje del diagnóstico correcto. Estos trastornos tienen graves consecuencias sobre el intercambio gaseoso, que junto con otras anomalías de la función pulmonar producen los signos y síntomas y afectan la calidad de vida. La utilización de parámetros fisiológicos no sólo ayuda en el diagnóstico, sino que además evalúan la gravedad, ayudan a definir consecuencias del tratamiento y son útiles durante el seguimiento. Algunas de las pruebas de función pulmonar puede ser completamente normales con enfermedad radiográficamente severa, pero 10% de los pacientes presentan alteraciones de la función pulmonar antes de los cambios radiológicos (1). La TACAR es una herramienta imagenológica esencial en el estudio de estos pacientes, no sólo en el diagnóstico, sino en parámetros clínicos como seguimiento mientras continua abriéndose camino en su utilidad pronostica. A continuación evaluaremos el uso clínico de la función pulmonar y la TACAR en las enfermedades pulmonares intersticiales.

Regulation of igaA and the Rcs system by the MviA response regulator in Salmonella enterica.

IgaA is a membrane protein that prevents overactivation of the Rcs regulatory system in enteric bacteria. Here we provide evidence that igaA is the first gene in a sigma(70)-dependent operon of Salmonella enterica serovar Typhimurium that also includes yrfG, yrfH, and yrfI. We also show that the Lon protease and the MviA response regulator participate in regulation of the igaA operon. Our results indicate that MviA regulates igaA transcription in an RpoS-dependent manner, but the results also suggest that MviA may regulate RcsB activation in an RpoS- and IgaA-independent manner.

Eosinófilos en sangre y en esputo como predictores de severidad en crisis asmática / Eosinophils in blood or sputum. predictors of severity in asthma attack

Algunos estudios sugieren que los eosinófilos se han correlacionado con mayor severidad, peor control del asma, peores índices de función pulmonar, mayor hiperreactividad bronquial medida con test de metacolina. No obstante, desconocemos el comportamiento de esta correlación en Colombia y en la clínica de asma del Hospital San Ignacio. Actualmente el wright de esputo se interpreta solo a la luz de la presencia de neutrófilos ignorando el valor de los eosinófilos. Métodos: Se realizó un estudio observacional descriptivo y de concordancia, durante un año, en personas mayores de 15 años, que ingresaron por urgencias con crisis asmática requirieron hospitalización. Se determinó en ellos la severidad de la crisis, la severidad de los eosinófilos en sangre y en esputo, el pico espiratorio flujo, los días de estancia totales, en piso, o en UCI y la necesidad de ventilación mecánica para definir si había correlación entre las severidades de estas variables como predictores de severidad de la crisis asmática. Resultados: Se incluyeron un total de 169 pacientes, con promedio de edad de 43.3 años, predominantemente mujeres (84 por ciento) de los cuales el 56.8 por ciento tenían crisis asmática severa y 43.2 por ciento moderada. Con un promedio de eosinófilos en esputo de 17.2 por ciento y en sangre de 375.5 células por metro cúbico. Promedio de estancia hospitalaria total de 4.7 días, 0.5 días en UCI (solamente 20 pacientes requirieron UCI), y 0.2 días en ventilación mecánica. La mayoría de pacientes tenían eosinófilos en sangre dentro de los valores normales o levemente aumentados y la mayoría del grupo tenía eosinofilia leve en esputo. El PEF al ingreso correspondía en promedio a un 40 por ciento del mejor. Solamente se observó concordancia leve entre eosinófilos en esputo y eosinófilos en sangre. No se encontró correlación entre severidad según eosinófilos en esputo o en sangre y los días de estancia, el PEF ni con las drogas recibidas. Conclusión: No existe co...

Rcs and PhoPQ regulatory overlap in the control of Salmonella enterica virulence.

Genetic screens based on the use of MudJ-generated lac fusions permitted the identification of novel genes regulated by the Rcs signal transduction system in Salmonella enterica serovar Typhimurium. Besides genes that are also found in the Escherichia coli genome, our screens identified Salmonella-specific genes regulated by RcsB, including bapA, siiE, srfA, and srfB. Here we show that the srfABC operon is negatively regulated by RcsB and by PhoP. In vivo studies using mutants with constitutive activation of the Rcs and/or PhoPQ system suggested that there is an overlap between these regulatory systems in the control of Salmonella virulence.

Virulence attenuation in Salmonella enterica rcsC mutants with constitutive activation of the Rcs system.

Mutations in rcsC that result in constitutive colanic acid capsule synthesis were obtained in Salmonella enterica serovar Typhimurium. Most rcsC alleles were dominant; however, recessive rcsC alleles were also found, in agreement with the postulated double role (positive and negative) of RcsC on the activation of the RcsB/C phosphorelay system. Salmonella rcsC mutants with constitutive activation of the Rcs system are severely attenuated for virulence in BALB/c mice and their degree of attenuation correlates with the level of Rcs activation. Partial relief of attenuation by a gmm mutation indicates that capsule overproduction is one of the factors leading to avirulence in constitutively activated rcsC mutants.